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Onset of streptococcal toxic shock syndrome is accelerated by bruising in a mouse model
ARTICLE IN PRESS
MICROBIAL PATHOGENESIS Microbial Pathogenesis 44 (2008) 339–343 www.elsevier.com/locate/micpath
Onset of streptococcal toxic shock syndrome is accelerated by bruising in a mouse model Masanori Sekia, Mitsumasa Saitoa,b, Ken-Ichiro Iidaa, Hiroaki Taniaia, Takashi Soejimaa, Hiroaki Nakayamaa, Shin-Ichi Yoshidaa, a
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan b Department of Pediatrics, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Received 10 September 2007; received in revised form 18 October 2007; accepted 18 October 2007 Available online 30 October 2007
Abstract Streptococcal toxic shock syndrome (STSS) is the severest form of human infections caused by Streptococcus pyogenes. In our animal model of STSS [Saito M, Kajiwara H, Ishikawa T, et al. Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes. Microbiol Immunol 2001;45:777–86], mice inoculated intramuscularly with S. pyogenes strains initially suffer from a short illness, then recover and remain healthy for about 20 days, and finally become sick and incur a sudden death. Here we report that the death during the convalescent period was facilitated by artificially bruising an extremity remote from the site of the initial inoculation. Bacterial burden was found to be higher in the bruised site than in a non-bruised control extremity of each mouse examined. Bacteremia started to occur approximately 20 days after infection. These findings imply that a fresh bruise serves as a focus for bacterial multiplication in the presence of bacteremia, thereby facilitating the development of STSS. r 2007 Elsevier Ltd. All rights reserved. Keywords: Streptococcus pyogenes; Streptococcal toxic shock syndrome; Bruise
1. Introduction Streptococcus pyogenes is an important human pathogen causing not only various pyogenic infections but also poststreptococcal diseases, i.e., glomerulonephritis and rheumatic fever. In the late 1980s, a severe invasive type of S. pyogenes infection characterized by septic shock often associated with cellulitis, necrotizing fasciitis and myositis began to attract considerable attention [1–5]. In 1993, the name and definition of streptococcal toxic shock syndrome (STSS) were proposed for this type of infection [6], but mechanisms for its development still remain unclear [7–9]. One of the reasons for the paucity of our understanding about STSS is due to the absence of a suitable animal model. In typical cases of human STSS, healthy people suddenly present overt symptoms and die within 48 h after onset, while the initial site of infection is usually unclear. Corresponding author. Tel.: +81 92 642 6127; fax: +81 92 642 6133.
E-mail address: [email protected] (S.-I. Yoshida). 0882-4010/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2007.10.009
This means that typical STSS does not represent a sequela of an acute infection. Animal studies thus far reported have not been successful in reproducing such features of human STSS. Thus, inoculating mice with S. pyogenes causes an acute disease, which is quickly followed by the death of the animals [7,9–14]. Obviously, such experimental infections do not mimic human STSS. We have previously reported a mouse model that is considered to simulate human STSS [15]. After surviving an acute infection, infected mice become apparently healthy, gain body weight, then suddenly turn sick, and soon die. Furthermore, some of the mice develop softtissue necrosis resembling necrotizing fasciitis in human STSS [16,17]. We called the death occurring 20 or more days after infection as delayed death and showed that the capsule is essential for the organism to cause delayed death in mice because hasA mutants are devoid of this ability [18]. S. pyogenes generally invades the host via the upper respiratory tract or injured skin [8]. A number of factors including varicella [19,20], penetrating injuries, burns,
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splinters, and childbirth have been assumed to predispose the host to the development of STSS [21]. Of particular interest is the observation that STSS occurred following a bruise in the absence of an open wound [22,23]. In the present study, we looked at the effect of an artificial bruise on the course of disease in our mouse model of STSS and found that it actually accelerated the mortality. 2. Results 2.1. Effects of artificial bruising on the mortality of infected mice
100 80 60 40
bruise
The fact that a bruise given at a location remote from the site of inoculation accelerated the death of the animals suggested a crucial role of bacteremia. To see if this supposition is actually the case, we looked at the time course of bacteremia. Blood samples were collected from
100 80 60 40
bruise
20
20 0
2.2. Occurrence of bacteremia
Survival rate (%)
Survival rate (%)
Cells of S. pyogenes SP2 were injected into the right forelimbs of 40 mice intramuscularly at a dose of 1 107 CFU per animal, and the inoculated mice were divided into two equal groups. Each mouse in one group was given an artificial bruise in the right hind leg by pinching with forceps 20 days after inoculation, while the other group served as the unbruised control. The control mice started to die 25 days after infection with about a half still surviving
40 days after infection. The bruised mice started to die from 20 days after the injury with more than 80% being dead by the 40th day of infection (Fig. 1A). Obviously, death, which we regard as the cardinal sign of murine STSS [15], occurred earlier in the bruised mice than in the control mice (Po0.005 by log-rank test). Furthermore, almost all of the bruised mice showed swelling in the bruised sites and some had a ruptured abscess as early as 3 days after being bruised (Fig. 2). It was also noted that when the artificial bruising was given 10 days after infection, there was no difference in the time course of mortality between the bruised and control groups (P40.05) (Fig. 1B).
10 20 30 Days after infection
40
0
10 20 30 Days after infection
40
Fig. 1. Effects of artificial bruising on the death of mice inoculated with S. pyogenes SP2. Forty (A) and 20 (B) mice were inoculated intramuscularly with 1 107 SP2 cells in the right forelimb. A half of them were bruised on the right hind legs by pinching 20 days (A) or 10 days (B) after infection (E) while the other half served as the non-bruised control (J). Log-rank test showed that the difference in survival days and survival rate between the bruised and non-bruised groups was statistically significant (Po0.005) in (A), but not in (B). The experiments were repeated three times under the same conditions with similar results. Representative results are shown in the figure.
Fig. 2. A ruptured abscess seen at the bruised site. The mouse was infected intramuscularly with 1 107 SP2 cells in the right forelimb and bruised on the right hind legs by pinching 20 after the infection. Abscess could be seen from 3 days after bruising: (A) a side view and (B) a view showing that the unbruised leg is free from an abscess.
ARTICLE IN PRESS M. Seki et al. / Microbial Pathogenesis 44 (2008) 339–343
Table 1 The bacterial burden in the bruised (20 days after infection) and nonbruised leg of infected mice
1 2 3
Mice no.
4 Mouse
341
5
Days until death
6 7 9 10 0
10
20 Days after infection
30
40
Fig. 3. Time course of bacteremia and mortality. Blood samples (0.1 ml) obtained at intervals from infected mice were examined for b-hemolytic colonies on blood-supplemented BHI agar plates. Open and closed circles denote negative and positive blood cultures, respectively; daggers signify deaths of animals.
10 infected mice at intervals and examined for the presence of b-hemolytic streptococci. The results showed that bacteremia became detectable on the 20th day of infection (Fig. 3), which roughly coincided with the onset of death. This finding seems to suggest a causal linkage between bacteremia and STSS. 2.3. Bacterial accumulation in the bruised leg Having found the acceleration of STSS onset by artificial bruising, we hypothesized that the bruised tissue became deficient in local defense and served as a bacterial amplifier. To test this hypothesis, we counted the number of viable S. pyogenes cells in bruised and non-bruised legs of seven dead mice (Table 1). The results showed that the bacterial burden was significantly larger in the bruised leg than in the non-bruised leg of each mouse examined (P ¼ 0.006 by paired t-test). The statistical difference was observed in a series of experiments. The result lends support to the above hypothesis. 2.4. Prevention of STSS by surgical intervention The above results strongly suggested that bacteremia was required for the development of STSS. This led us to suppose that the removal of the site of the initial infection before the emergence of bacteremia could effectively prevent the onset of STSS. A total of 20 mice were infected with 1 107 CFU of SP2 strain into right foreleg. In the half of mice, the infected leg was amputated 10 days after the inoculation. When the infected leg was amputated, 80% of the mice remained alive for 70 days, whereas only 20% of the control mice without this intervention survived (Fig. 4). The difference in survival days and survival rate between the two groups was statistically significant (P ¼ 0.01) by log-rank test.
1 2 3 4 5 6 7
24 26 30 32 33 37 38
Mean7SD
Difference
Right (bruised)
Left (non-bruised)
7.35 7.36 6.60 7.30 5.80 5.48 5.48
4.62 3.48 4.88 4.70 4.85 4.72 4.72
2.93 3.88 1.72 2.60 0.95 0.76 0.76
6.5470.96
4.5170.49
1.9771.25
100
Survival rate (%)
8
Bacterial numbers (log10 CFU) in legs
80 60 40 20
0 10
20
30 40 50 Days after infection
60
Fig. 4. Effects of removal of the initial infection site on the mortality. Twenty mice were inoculated as described, 10 of which underwent amputation of the infected right forelegs 10 days after infection (J), with the rest serving as control (E). The differences in survival rate and survival days between the amputated (J) and non-amputated (E) groups were statistically significant (P ¼ 0.01) by log-rank test.
3. Discussion Our previous report described an experimental infection in which mice inoculated with S. pyogenes died a sudden death with bacteremia after incubation periods of at least 3 weeks in length [15]. We pointed out that the ‘‘delayed death’’ in this experimental infection was similar to human STSS in the following aspects. First, the death is preceded by a long incubation period of apparently good health. Second, approximately 20% of the infected mice developed soft-tissue necrosis. Because of these features, we consider that our system qualifies as a mouse model of human STSS. In a typical case of human STSS, a minor injury is often implicated in the disease. Furthermore, there are reports documenting that even a bruise with no accompanying skin injury resulted in STSS [22,23]. Our present results seem to
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be consistent with those clinical observations. Thus, an artificial bruise made 20 days after infection clearly accelerated the onset of death, which strongly support the view that the induction of STSS is facilitated by the presence of a bruise. Interestingly, this accelerating effect was absent if bruising was made 10 days after infection. This discrepancy may be explained as follows. Bacteremia was shown to start approximately 20 days after infection. Then, it can be postulated that if bacteremia occurs in the presence of a fresh bruise, i.e., one given on the 20th day of infection, bacteria reach the bruised site, which is suitable for bacterial growth due to deteriolated local resistance (so-called locus minoris resistantiae), and multiply rapidly there, thus aggravating bacteremia and eventually resulting in STSS. In the case of early bruising made on the 10th day of infection, the bruise has already healed sufficiently to regain the normal level of resistance when bacteremia begins. This view is also consistent with the finding that the bruised leg has a heavier burden of bacteria than an unbruised leg. The development of STSS was effectively avoided by surgical removal of the initial site of infection prior to the emergence of bacteremia. This seems to support the clinical view stressing the importance of preventing bacteremia in avoiding the onset of STSS [17,24]. In conclusion, our results obtained by using our mouse model of STSS indicate that a bruise serves as a bacterial amplifier in the presence of bacteremia, thereby accelerating the onset of the disease. 4. Materials and methods 4.1. Bacterial strain and culture media The S. pyogenes strain used in this study was SP2, originally isolated from an STSS patient (M type unknown, T type 11, SpeA negative, and SpeB positive). Brain heart infusion (BHI) broth (Eiken Chemical, Tokyo) and BHI agar (Eiken Chemical) supplemented with 10% sheep blood were used as the liquid and solid media, respectively.
tenth milliliter of the bacterial suspension containing 1 107 CFU was injected intramuscularly into the right foreleg of each mouse. Artificial bruising was given by pinching the right hind leg with forceps. When needed, the initial site of infection was removed by amputating the right foreleg where the inoculation had been made. All these operations were carried out under anesthesia induced by administering pentobarbital sodium at a dose of 0.075 mg/g body weight via the tail vein. The experimental protocol was reviewed by the Committee of Ethics on Animal Experiments of the Faculty of Medical Sciences, Kyushu University, and carried out in accordance with the Animal Experiment Guidelines of the Faculty and the Law No. 105 and the Notification No. 6 of the Japanese Government. 4.4. Detection and count of bacteria in blood and tissues Blood samples (0.1 ml) were collected at intervals from the tail vein of infected mice and spread on sheep bloodsupplemented BHI agar plates. The plates were incubated aerobically in the presence of 5% CO2 at 37 1C, and b-hemolytic colonies were counted after 18 h. Legs were cut off from infected mice (n=7) just after death and freed from the skin and bones as much as possible. The mass consisting mainly of muscles was placed in a Teflon homogenizer containing 2 ml of sterile PBS and homogenized. The homogenates were serially diluted with PBS, and 0.1 ml of each dilution was spread on sheep bloodsupplemented BHI agar plates, which were incubated at 37 1C for 18 h. Limit of detection is 20 CFU/leg muscle. 4.5. Statistics Kaplan–Meier survival analysis was applied to verify the effect of artificial bruising (Fig. 1) or amputation of infected sites (Fig. 4) and the statistical differences were evaluated by the log-rank test. Difference in the CFU (Table 1) was tested by paired t-test. Acknowledgements
4.2. Bacterial inocula Exponential-phase cells of strain SP2 grown in BHI broth at 37 1C with shaking were harvested by low-speed centrifugation. The cells were washed twice with, and resuspended in, sterile phosphate-buffered saline (PBS) at a density of 1 108 CFU/ml. The cell density was routinely adjusted by measuring the turbidity at 660 nm, which had been calibrated against CFU determined by plating on the blood-supplemented BHI agar medium. 4.3. Mouse experiments Six-week-old male mice of ddY strain, purchased from Kyudo Co. Ltd., Kumamoto, Japan, were used. The one-
We thank Hideko Kajiwara for her technical assistance. We also thank Sharon Y.A.M Villanueva for her editorial advice on the manuscript. This work was supported by the Grant-in-aid from Ministry of Education, Culture, Sports and Technology no. 18790310. References [1] Demers B, Simor AE, Vellend H, et al. Severe invasive group A streptococcal infections in Ontario, Canada: 1987–1991. Clin Infect Dis 1993;16:792–800 discussion 801–2. [2] Hoge CW, Schwartz B, Talkington DF, et al. The changing epidemiology of invasive group A streptococcal infections and the emergence of streptococcal toxic shock-like syndrome. A retrospective population-based study. JAMA 1993;269:384–9.
ARTICLE IN PRESS M. Seki et al. / Microbial Pathogenesis 44 (2008) 339–343 [3] Martin PR, Hoiby EA. Streptococcal serogroup A epidemic in Norway 1987–1988. Scand J Infect Dis 1990;22:421–9. [4] Stevens DL, Tanner MH, Winship J, et al. Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 1989;321:1–7. [5] Stevens DL. Invasive group A streptococcus infections. Clin Infect Dis 1992;14:2–11. [6] The Working Group on Severe Streptococcal Infections. Defining the group A streptococcal toxic shock syndrome. Rationale and consensus definition. JAMA 1993;269:390–1. [7] Ashbaugh CD, Warren HB, Carey VJ, Wessels MR. Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection. J Clin Invest 1998;102:550–60. [8] Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470–511. [9] Raeder R, Boyle MD. Association between expression of immunoglobulin G-binding proteins by group A streptococci and virulence in a mouse skin infection model. Infect Immun 1993;61:1378–84. [10] Boyle MD, Raeder R, Flosdorff A, Podbielski A. Role of emm and mrp genes in the virulence of group A streptococcal isolate 64/14 in a mouse model of skin infection. J Infect Dis 1998;177:991–7. [11] Okamoto S, Kawabata S, Nakagawa I, et al. Influenza A virusinfected hosts boost an invasive type of Streptococcus pyogenes infection in mice. J Virol 2003;77:4104–12. [12] Schmidt KH, Podbielski A, Raeder R, Boyle MD. Inactivation of single genes within the virulence regulon of an M2 group A streptococcal isolate result in differences in virulence for chicken embryos and for mice. Microb Pathog 1997;23:347–55. [13] Shiseki M, Miwa K, Nemoto Y, et al. Comparison of pathogenic factors expressed by group A streptococci isolated from patients with streptococcal toxic shock syndrome and scarlet fever. Microb Pathog 1999;27:243–52.
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[14] Sriskandan S, Unnikrishnan M, Krausz T, Cohen J. Molecular analysis of the role of streptococcal pyrogenic Exotoxin A (SPEA) in invasive soft-tissue infection resulting from Streptococcus pyogenes. Mol Microbiol 1999;33:778–90. [15] Saito M, Kajiwara H, Ishikawa T, et al. Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes strains. Microbiol Immunol 2001;45:777–86. [16] Stamenkovic I, Lew PD. Early recognition of potentially fatal necrotizing fasciitis. The use of frozen-section biopsy. N Engl J Med 1984;310:1689–93. [17] Wood TF, Potter MA, Jonasson O. Streptococcal toxic shock-like syndrome. The importance of surgical intervention. Ann Surg 1993;217:109–14. [18] Iida K, Seki M, Saito M, et al. Capsule of Streptococcus pyogenes is essential for delayed death of mice in a model of streptococcal toxic shock syndrome. Microbiol Immunol 2006;50:127–30. [19] Cowan MR, Primm PA, Scott SM, et al. Serious group A betahemolytic streptococcal infections complicating varicella. Ann Emerg Med 1994;23:818–22. [20] Doctor A, Harper MB, Fleisher GR. Group A beta-hemolytic streptococcal bacteremia: historical overview, changing incidence, and recent association with varicella. Pediatrics 1995;96:428–33. [21] Bisno AL, Stevens DL. Streptococcal infections of skin and soft tissues. N Engl J Med 1996;334:240–5. [22] Gentry RH, Fitzpatrick JE. A peculiar purple bruise. Necrotizing fasciitis due to a group-A beta-hemolytic streptococci. Arch Dermatol 1990;126:816–7. 819–20. [23] Hirose Y, Shibuya H, Okazaki E, et al. Toxic shock-like syndrome with flu-like prodrome: a possible role of ‘enhancing tissue focus’ for streptococcal toxic shock. J Infect 2001;42:195–200. [24] Zittergruen M, Grose C. Magnetic resonance imaging for early diagnosis of necrotizing fasciitis. Pediatr Emerg Care 1993;9:26–8.
MICROBIAL PATHOGENESIS Microbial Pathogenesis 44 (2008) 339–343 www.elsevier.com/locate/micpath
Onset of streptococcal toxic shock syndrome is accelerated by bruising in a mouse model Masanori Sekia, Mitsumasa Saitoa,b, Ken-Ichiro Iidaa, Hiroaki Taniaia, Takashi Soejimaa, Hiroaki Nakayamaa, Shin-Ichi Yoshidaa, a
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan b Department of Pediatrics, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Received 10 September 2007; received in revised form 18 October 2007; accepted 18 October 2007 Available online 30 October 2007
Abstract Streptococcal toxic shock syndrome (STSS) is the severest form of human infections caused by Streptococcus pyogenes. In our animal model of STSS [Saito M, Kajiwara H, Ishikawa T, et al. Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes. Microbiol Immunol 2001;45:777–86], mice inoculated intramuscularly with S. pyogenes strains initially suffer from a short illness, then recover and remain healthy for about 20 days, and finally become sick and incur a sudden death. Here we report that the death during the convalescent period was facilitated by artificially bruising an extremity remote from the site of the initial inoculation. Bacterial burden was found to be higher in the bruised site than in a non-bruised control extremity of each mouse examined. Bacteremia started to occur approximately 20 days after infection. These findings imply that a fresh bruise serves as a focus for bacterial multiplication in the presence of bacteremia, thereby facilitating the development of STSS. r 2007 Elsevier Ltd. All rights reserved. Keywords: Streptococcus pyogenes; Streptococcal toxic shock syndrome; Bruise
1. Introduction Streptococcus pyogenes is an important human pathogen causing not only various pyogenic infections but also poststreptococcal diseases, i.e., glomerulonephritis and rheumatic fever. In the late 1980s, a severe invasive type of S. pyogenes infection characterized by septic shock often associated with cellulitis, necrotizing fasciitis and myositis began to attract considerable attention [1–5]. In 1993, the name and definition of streptococcal toxic shock syndrome (STSS) were proposed for this type of infection [6], but mechanisms for its development still remain unclear [7–9]. One of the reasons for the paucity of our understanding about STSS is due to the absence of a suitable animal model. In typical cases of human STSS, healthy people suddenly present overt symptoms and die within 48 h after onset, while the initial site of infection is usually unclear. Corresponding author. Tel.: +81 92 642 6127; fax: +81 92 642 6133.
E-mail address: [email protected] (S.-I. Yoshida). 0882-4010/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2007.10.009
This means that typical STSS does not represent a sequela of an acute infection. Animal studies thus far reported have not been successful in reproducing such features of human STSS. Thus, inoculating mice with S. pyogenes causes an acute disease, which is quickly followed by the death of the animals [7,9–14]. Obviously, such experimental infections do not mimic human STSS. We have previously reported a mouse model that is considered to simulate human STSS [15]. After surviving an acute infection, infected mice become apparently healthy, gain body weight, then suddenly turn sick, and soon die. Furthermore, some of the mice develop softtissue necrosis resembling necrotizing fasciitis in human STSS [16,17]. We called the death occurring 20 or more days after infection as delayed death and showed that the capsule is essential for the organism to cause delayed death in mice because hasA mutants are devoid of this ability [18]. S. pyogenes generally invades the host via the upper respiratory tract or injured skin [8]. A number of factors including varicella [19,20], penetrating injuries, burns,
ARTICLE IN PRESS M. Seki et al. / Microbial Pathogenesis 44 (2008) 339–343
340
splinters, and childbirth have been assumed to predispose the host to the development of STSS [21]. Of particular interest is the observation that STSS occurred following a bruise in the absence of an open wound [22,23]. In the present study, we looked at the effect of an artificial bruise on the course of disease in our mouse model of STSS and found that it actually accelerated the mortality. 2. Results 2.1. Effects of artificial bruising on the mortality of infected mice
100 80 60 40
bruise
The fact that a bruise given at a location remote from the site of inoculation accelerated the death of the animals suggested a crucial role of bacteremia. To see if this supposition is actually the case, we looked at the time course of bacteremia. Blood samples were collected from
100 80 60 40
bruise
20
20 0
2.2. Occurrence of bacteremia
Survival rate (%)
Survival rate (%)
Cells of S. pyogenes SP2 were injected into the right forelimbs of 40 mice intramuscularly at a dose of 1 107 CFU per animal, and the inoculated mice were divided into two equal groups. Each mouse in one group was given an artificial bruise in the right hind leg by pinching with forceps 20 days after inoculation, while the other group served as the unbruised control. The control mice started to die 25 days after infection with about a half still surviving
40 days after infection. The bruised mice started to die from 20 days after the injury with more than 80% being dead by the 40th day of infection (Fig. 1A). Obviously, death, which we regard as the cardinal sign of murine STSS [15], occurred earlier in the bruised mice than in the control mice (Po0.005 by log-rank test). Furthermore, almost all of the bruised mice showed swelling in the bruised sites and some had a ruptured abscess as early as 3 days after being bruised (Fig. 2). It was also noted that when the artificial bruising was given 10 days after infection, there was no difference in the time course of mortality between the bruised and control groups (P40.05) (Fig. 1B).
10 20 30 Days after infection
40
0
10 20 30 Days after infection
40
Fig. 1. Effects of artificial bruising on the death of mice inoculated with S. pyogenes SP2. Forty (A) and 20 (B) mice were inoculated intramuscularly with 1 107 SP2 cells in the right forelimb. A half of them were bruised on the right hind legs by pinching 20 days (A) or 10 days (B) after infection (E) while the other half served as the non-bruised control (J). Log-rank test showed that the difference in survival days and survival rate between the bruised and non-bruised groups was statistically significant (Po0.005) in (A), but not in (B). The experiments were repeated three times under the same conditions with similar results. Representative results are shown in the figure.
Fig. 2. A ruptured abscess seen at the bruised site. The mouse was infected intramuscularly with 1 107 SP2 cells in the right forelimb and bruised on the right hind legs by pinching 20 after the infection. Abscess could be seen from 3 days after bruising: (A) a side view and (B) a view showing that the unbruised leg is free from an abscess.
ARTICLE IN PRESS M. Seki et al. / Microbial Pathogenesis 44 (2008) 339–343
Table 1 The bacterial burden in the bruised (20 days after infection) and nonbruised leg of infected mice
1 2 3
Mice no.
4 Mouse
341
5
Days until death
6 7 9 10 0
10
20 Days after infection
30
40
Fig. 3. Time course of bacteremia and mortality. Blood samples (0.1 ml) obtained at intervals from infected mice were examined for b-hemolytic colonies on blood-supplemented BHI agar plates. Open and closed circles denote negative and positive blood cultures, respectively; daggers signify deaths of animals.
10 infected mice at intervals and examined for the presence of b-hemolytic streptococci. The results showed that bacteremia became detectable on the 20th day of infection (Fig. 3), which roughly coincided with the onset of death. This finding seems to suggest a causal linkage between bacteremia and STSS. 2.3. Bacterial accumulation in the bruised leg Having found the acceleration of STSS onset by artificial bruising, we hypothesized that the bruised tissue became deficient in local defense and served as a bacterial amplifier. To test this hypothesis, we counted the number of viable S. pyogenes cells in bruised and non-bruised legs of seven dead mice (Table 1). The results showed that the bacterial burden was significantly larger in the bruised leg than in the non-bruised leg of each mouse examined (P ¼ 0.006 by paired t-test). The statistical difference was observed in a series of experiments. The result lends support to the above hypothesis. 2.4. Prevention of STSS by surgical intervention The above results strongly suggested that bacteremia was required for the development of STSS. This led us to suppose that the removal of the site of the initial infection before the emergence of bacteremia could effectively prevent the onset of STSS. A total of 20 mice were infected with 1 107 CFU of SP2 strain into right foreleg. In the half of mice, the infected leg was amputated 10 days after the inoculation. When the infected leg was amputated, 80% of the mice remained alive for 70 days, whereas only 20% of the control mice without this intervention survived (Fig. 4). The difference in survival days and survival rate between the two groups was statistically significant (P ¼ 0.01) by log-rank test.
1 2 3 4 5 6 7
24 26 30 32 33 37 38
Mean7SD
Difference
Right (bruised)
Left (non-bruised)
7.35 7.36 6.60 7.30 5.80 5.48 5.48
4.62 3.48 4.88 4.70 4.85 4.72 4.72
2.93 3.88 1.72 2.60 0.95 0.76 0.76
6.5470.96
4.5170.49
1.9771.25
100
Survival rate (%)
8
Bacterial numbers (log10 CFU) in legs
80 60 40 20
0 10
20
30 40 50 Days after infection
60
Fig. 4. Effects of removal of the initial infection site on the mortality. Twenty mice were inoculated as described, 10 of which underwent amputation of the infected right forelegs 10 days after infection (J), with the rest serving as control (E). The differences in survival rate and survival days between the amputated (J) and non-amputated (E) groups were statistically significant (P ¼ 0.01) by log-rank test.
3. Discussion Our previous report described an experimental infection in which mice inoculated with S. pyogenes died a sudden death with bacteremia after incubation periods of at least 3 weeks in length [15]. We pointed out that the ‘‘delayed death’’ in this experimental infection was similar to human STSS in the following aspects. First, the death is preceded by a long incubation period of apparently good health. Second, approximately 20% of the infected mice developed soft-tissue necrosis. Because of these features, we consider that our system qualifies as a mouse model of human STSS. In a typical case of human STSS, a minor injury is often implicated in the disease. Furthermore, there are reports documenting that even a bruise with no accompanying skin injury resulted in STSS [22,23]. Our present results seem to
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be consistent with those clinical observations. Thus, an artificial bruise made 20 days after infection clearly accelerated the onset of death, which strongly support the view that the induction of STSS is facilitated by the presence of a bruise. Interestingly, this accelerating effect was absent if bruising was made 10 days after infection. This discrepancy may be explained as follows. Bacteremia was shown to start approximately 20 days after infection. Then, it can be postulated that if bacteremia occurs in the presence of a fresh bruise, i.e., one given on the 20th day of infection, bacteria reach the bruised site, which is suitable for bacterial growth due to deteriolated local resistance (so-called locus minoris resistantiae), and multiply rapidly there, thus aggravating bacteremia and eventually resulting in STSS. In the case of early bruising made on the 10th day of infection, the bruise has already healed sufficiently to regain the normal level of resistance when bacteremia begins. This view is also consistent with the finding that the bruised leg has a heavier burden of bacteria than an unbruised leg. The development of STSS was effectively avoided by surgical removal of the initial site of infection prior to the emergence of bacteremia. This seems to support the clinical view stressing the importance of preventing bacteremia in avoiding the onset of STSS [17,24]. In conclusion, our results obtained by using our mouse model of STSS indicate that a bruise serves as a bacterial amplifier in the presence of bacteremia, thereby accelerating the onset of the disease. 4. Materials and methods 4.1. Bacterial strain and culture media The S. pyogenes strain used in this study was SP2, originally isolated from an STSS patient (M type unknown, T type 11, SpeA negative, and SpeB positive). Brain heart infusion (BHI) broth (Eiken Chemical, Tokyo) and BHI agar (Eiken Chemical) supplemented with 10% sheep blood were used as the liquid and solid media, respectively.
tenth milliliter of the bacterial suspension containing 1 107 CFU was injected intramuscularly into the right foreleg of each mouse. Artificial bruising was given by pinching the right hind leg with forceps. When needed, the initial site of infection was removed by amputating the right foreleg where the inoculation had been made. All these operations were carried out under anesthesia induced by administering pentobarbital sodium at a dose of 0.075 mg/g body weight via the tail vein. The experimental protocol was reviewed by the Committee of Ethics on Animal Experiments of the Faculty of Medical Sciences, Kyushu University, and carried out in accordance with the Animal Experiment Guidelines of the Faculty and the Law No. 105 and the Notification No. 6 of the Japanese Government. 4.4. Detection and count of bacteria in blood and tissues Blood samples (0.1 ml) were collected at intervals from the tail vein of infected mice and spread on sheep bloodsupplemented BHI agar plates. The plates were incubated aerobically in the presence of 5% CO2 at 37 1C, and b-hemolytic colonies were counted after 18 h. Legs were cut off from infected mice (n=7) just after death and freed from the skin and bones as much as possible. The mass consisting mainly of muscles was placed in a Teflon homogenizer containing 2 ml of sterile PBS and homogenized. The homogenates were serially diluted with PBS, and 0.1 ml of each dilution was spread on sheep bloodsupplemented BHI agar plates, which were incubated at 37 1C for 18 h. Limit of detection is 20 CFU/leg muscle. 4.5. Statistics Kaplan–Meier survival analysis was applied to verify the effect of artificial bruising (Fig. 1) or amputation of infected sites (Fig. 4) and the statistical differences were evaluated by the log-rank test. Difference in the CFU (Table 1) was tested by paired t-test. Acknowledgements
4.2. Bacterial inocula Exponential-phase cells of strain SP2 grown in BHI broth at 37 1C with shaking were harvested by low-speed centrifugation. The cells were washed twice with, and resuspended in, sterile phosphate-buffered saline (PBS) at a density of 1 108 CFU/ml. The cell density was routinely adjusted by measuring the turbidity at 660 nm, which had been calibrated against CFU determined by plating on the blood-supplemented BHI agar medium. 4.3. Mouse experiments Six-week-old male mice of ddY strain, purchased from Kyudo Co. Ltd., Kumamoto, Japan, were used. The one-
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